The present invention relates generally to delivery of oligonucleotides to targeted cell types, and is in particular a means using microparticulates having on their outer surface molecules which are specifically bound and taken up by cells expressing the heme receptor.
Targeted drug delivery is required in a number of situations where the drug may be toxic if administered in a very high dose in order for sufficient doses to reach an intended site, where the drug is very expensive or where the drug is subject to rapid removal or degradation. Drug delivery, and more recently delivery of oligonucleotides and genes, to targeted cells has been modulated by manipulation of carrier, method of manufacture, and attachment of targeting molecules. Although drugs are traditionally encapsulated in tablets or capsules for oral delivery, encapsulation into more sophisticated vehicles is required for targeted delivery and delivery of molecules such as oligonucleotides and genes, which are extremely sensitive to the presence of nucleases in the body.
Reports of delivery of oligonucleotides using liposomes and polymeric vehicles for local release have been made, although no device is publicly available at this time. Liposomes consist basically of a phospholipid bilayer forming a shell around an aqueous core. Advantages include the lipophilicity of the outer layers which "mimic" the outer membrane layers of cells and that they are taken up relatively easily by a variety of cells. Disadvantages include the non-specific nature of delivery. Polymeric vehicles typically consist of microspheres and microcapsules formed of biocompatible polymers, which are either biodegradable (for example, polylactic acid) or non-biodegradable (for example, ethylenevinyl acetate). Some of the advantages of the polymeric devices are ease of manufacture and high loading capacity, range of size from nanometer to micron diameter, as well as controlled release and degradation profile. However, targeting of the polymeric vehicles remains as problematic as with liposomes. Both liposomes and small polymeric vehicles are referred to herein as "microparticles", unless specifically stated otherwise.
Many different systems have been proposed for targeted drug delivery. The most commonly used method has been to covalently attach specific antibodies to the surface of microparticulate carriers. Few systems have been demonstrated to deliver nucleic acids efficiently.
The use of liposomes for delivery of various drugs, including nucleic acid-based therapeutics, has been reported by Thierry, A. T., et al., Cancer Comm., 1, 311-316 (1989); and Thierry, A. T., et al., Biochem. Biophys. Res. Comm., 190, 952-960 (1993). Liposomes are potentially useful because they protect the encapsulated material from degradation by serum enzymes. Also, liposomes offer a distinct benefit to the delivery of drugs to the liver because, depending on their lipid composition and size, up to 70% of liposomes injected intravenously into mice may quickly locate to the liver, as reported by Liu, D., et al., Biochim. Biophys. Acta, 1104, 95-101 (1992).
Hepatocytes contain surface receptors for molecules such as asialoglycoprotein or transferrin, and these proteins have been used as the basis for ligand-directed delivery of certain molecules to hepatocytes, described by Chowdhury, N. R., et al., J. Biol Chem. 268, 11265-11271 (1993).
Immunoliposomes, i.e., liposomes bearing antibodies to certain target molecules, have also been used to direct drug delivery. However, the coupling of such complex ligands to the, surface of liposomes presents a number of problems, mainly due to the fact that they have a polypeptide or carbohydrate component and therefore contain multiple reactive groups. Attempts to carry out carbodiimide-mediated peptide bond formation between such complex ligand molecules and primary amino groups on the outer surface of liposomes can result in considerable intramolecular coupling and intermolecular coupling between ligand molecules, in addition to the desired intermolecular coupling between liposome and ligand. Furthermore, this coupling is difficult to regulate because it may result in a significant number of undesirable ligand structures, decreasing efficiency of the reaction. Protein ligands are also immunogenic and could therefore either evoke an immune reaction or result in clearance by the immune system of the body.
It is therefore an object of the present invention to provide a means for targeting microparticles for delivery to specific cell types not using antibodies or other proteins.
It is a further object of the present invention to provide compositions for efficient and reliable delivery to specific cell types, especially of nucleic acid-type molecules, such as antisense and ribozymes.